Modelling and analysis of early aggregation events of BMHP1-derived self-assembling peptides

<div><p>Despite the increasing use and development of peptide-based scaffolds in different fields including that of regenerative medicine, the understanding of the factors governing the self-assembly process and the relationship between sequence and properties have not yet been fully understood. BMHP1-derived self-assembling peptides (SAPs) have been developed and characterized showing that biotinylation at the N-terminal cap corresponds to better performing assembly and scaffold biomechanics. In this study, the effects of biotinylation on the self-assembly dynamics of seven BMHP1-derived SAPs have been investigated by molecular dynamics simulations. We confirmed that these SAPs self-assemble into <i>β</i>-structures and that proline acts as a <i>β</i>-breaker of the assembled aggregates. In biotinylated peptides, the formation of ordered <i>β</i>-structured aggregates is triggered by both the establishment of a dense and dynamic H-bonds network and the formation of a ‘hydrophobic wall’ available to interact with other peptides. Such conditions result from the peculiar chemical composition of the biotinyl-cap, given by the synergic cooperation of the uracil function of the ureido ring with the high hydrophobic portion consisting of the thiophenyl ring and valeryl chain. The inbuilt propensity of biotinylated peptides towards the formation of ordered small aggregates makes them ideal precursors of higher hierarchically organized self-assembled nanostructures as experimentally observed.</p></div

Figure 5

<p>ALP Staining images after culturing on the different hydrogels for two weeks. The bar represents 100 µm. RAD-I∶RADA16 1% (w/v), ALKmx∶ALK 1% (w/v)+RADA16, DGRmx∶DGR 1% (w/v)+RAD, PRGmx∶PRG 1% (w/v)+RADA16 (all mixture ratio is 1∶1). The bluish color intensity correlates with the high ALP activity. RADA16 shows low cell adhesion to the hydrogel and the cells are aggregated. The cell attachment increases in DGR and PRG scaffolds were considered as a result of RGD cell attachment sequence. ALP, DGR and PRG showed higher ALP activities compared to RADA16-I, especially staining intensity of PRG.</p

Figure 1

<p>Molecular models of pure and designer peptide nanofibers. A) Models represent peptide RADA16, ALK, DGR and PRG from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000190#pone-0000190-t001" target="_blank">Table 1</a>. B) Model representing a β-sheet double-tape of a self-assembling peptide nanofiber with PRG motif (4∶1). Note the sequences PRG extending out from the nanofiber double-tape.</p

Figure 5

<p>Images of neural stem cells in differentiation assays. a–d) Differentiating adult mouse neural stem cells cultured for 7 days <i>in vitro</i> on (a) 1% Matrigel, (b) RADA16 peptide scaffold; (c) RADA16-BMHP1 and (d) RADA16-BMHP2, 1% peptide scaffolds were examined with staining assays for DAPI (cell nuclei in blue), β-Tubulin⁺ (neurons in red), and Nestin⁺ (neural progenitors in green). β-Tubulin⁺ cells on BMHP1 and BMHP2 showed increased branching in comparison with RADA16 scaffold. These appearances are comparable with neurons on Matrigel coated wells. Nestin⁺ and β-Tubulin⁺ signals show negligible cross-reaction (MERGE images).</p

Figure 4

<p>MTT cell proliferation assays of adult mouse neural stem cells after 7-day culture. Cells were seeded on Matrigel or peptide scaffolds. The results are expressed as cell % increases from the seeding population on first day. BMHP1 and BMHP2 peptide scaffolds allow for higher cell proliferation in comparison to RADA16 peptide scaffold (t = −7.28 and t = −5.28 for respectively RADA16 vs. RADA16-BMHP1 and RADA16 vs. RADA16-BMHP2 with p<0.0001% in both cases). Not surprisingly, Matrigel containing various unknown quantity of growth factors showed considerable cell population increase. Similar increases of total cell populations were confirmed for 14-day cultures (not shown). The cell proliferation using the designer peptide scaffolds could be further improved from addition of soluble neurotrophic factors.</p

Figure 3

<p>SEM images of adult mouse neural stem cells (NSC) embedded in designer peptide nanofiber scaffold RADA16-BMHP1 (1% v/w) after 14 day <i>in vitro</i> cultures. I) Cluster of three visible NSCs (white circle) embedded in 3-D self-assembling RADA16-BMHP1. II) A single cell at different magnification with extended processes embedded in the scaffold is shown (a–c). White arrows point to the image areas enlarged in the consecutive pictures. d) High-magnification picture focusing on the interface between the nanofiber scaffold and the round shaped cell body. The black arrow in (b) points to a cellular process. Cells and processes are thus embedded in the self-assembling peptide nanofiber scaffold in a true 3-D environment, which may likely promote cell adhesions in 3-D similar to the natural cellular environment. Adult mouse neural stem cells have been cultured and could be differentiated <i>in vitro</i> for several weeks. The scale bars are shown on each image.</p

Figure 6

<p>Quantitative cell differentiation assays. The adult mouse neural stem cells were stained with various markers 7 days after culturing on Matrigel (positive control), RADA16, RADA16-BMHP1 and RADA16-BMHP2 peptide scaffolds. Values are expressed as averages±STD. a) % Nestin⁺ progenitor cell was reduced on the cultures of Matrigel, RADA16-BMHP1 and RADA16-BMHP2 peptide scaffolds, suggesting cell differentiation. b) % β-Tubulin⁺ cell was similar for Matrigel culture, RADA16-BMHP1 and RADA16-BMHP2 peptide scaffolds, suggesting neuronal differentiation. c) % GFAP⁺ cell was slightly higher in Matrigel culture, RADA16-BMHP1 and RADA16-BMHP2 peptide scaffolds, suggesting the ability of glial cells to adapt to a wide range of substrates.</p

Figure 3

<p>Cell numbers are calculated from DNA measurement on various scaffolds. MT3T3-E1 cells were cultured for 2 weeks on different scaffolds. RADA16∶RADA16 1% (w/v), ALKmx∶ALK 1% (w/v)+RADA16, DGRmx∶DGR 1% (w/v)+RADA16, PRGmx: PRG 1% (w/v)+RADA16 (all mix ratio is 1∶1). For peptide scaffolds containing active peptides, the cell proliferation rate is higher than that of pure RADA16. *p<0.01; suggesting it is significant against the number of cells grown in pure RADA16 scaffold.</p

Functionalized peptide scaffolds used in this study

<p>The sequences are from N–>C. Ac  = acetylated N-termini, −CONH₂ = amidated C-termini. The peptide motif sources from various protein origins. The 2-unit RGD motifs are purely molecular designed.</p

Figure 7

<p>Cell morphology on the different scaffolds of various mix ratios of RADA16 1% (w/v) and PRG 1% (w/v) using calcein-AM staining. The bar represents 100 µm. A) RADA16 100%∶PRG 0%, B) PRG 1%, C) PRG 10%, D) PRG 40%, E) PRG 70%, F) PRG 100% (RADA16: 0%). B) PRG: 1% shows uniform cell distribution compared to (A) 0%, which shows the increase of cell attachment. There is significant morphological difference in (C) 10% and (D) 40%, as seen by a change in cell shape from elongated form to asteroid form.</p